Oligosaccharide ingredient

ABSTRACT

An oligosaccharide ingredient comprising glycosylated amino acids and peptides of the general formula RnSacm where R is an amino acid residue, Sac is a monosaccharide selected from the group comprising N-acetyl-neuraminic acid, N-acetyl galactosamine and galactose, n has a value between 1 and 10 with the proviso that if n has the value 1 R is a threonine residue or a serine residue and if n has a value between 2 and 10 the peptide contains at least one threonine or serine residue, m has a value between 2 and 4 and at least 20 mol % of the ingredient is N-acetyl-neuraminic acid.

FIELD OF THE INVENTION

The invention relates to an oligosaccharide ingredient rich in sialicacid, food products comprising said oligosaccharide ingredient andprocesses for producing said oligosaccharide ingredient.

BACKGROUND OF THE INVENTION

Human milk is known to contain a large amount of indigestibleoligosaccharides. In fact, indigestible oligosaccharides represent thethird largest solid component (after lactose and lipids) in breast milk,occurring at a concentration of 12-15 g/l in colostrum and 5-8 g/l inmature milk. Human milk oligosaccharides are very resistant to enzymatichydrolysis, indicating that these oligosaccharides may display essentialfunctions not directly related to their calorific value.

Over the past two decades, the chemical structures of human milkoligosaccharides have been studied using NMR spectroscopy and massspectrometry. Each individual oligosaccharide is based on a combinationof glucose, galactose, sialic acid (N-acetylneuraminic acid), fucoseand/or N-acetylglucosamine with many and varied linkages between them,thus accounting for the enormous number of different oligosaccharides inhuman milk—over 130 such structures have been identified so far. Almostall of them have a lactose moiety at their reducing end while sialicacid (when present) occupies the terminal position at the non-reducingend.

The large quantity of sialylated oligosaccharides in human milk is ofparticular interest. Sialic acid is a nine-C sugar that is a vitalstructural and functional component of brain gangliosides. It is thoughtto play an essential role in nerve cell transmission, memory formationand cell to cell communication. Studies in rat pups indicate that earlysupplementation with sialic acid improves both brain ganglioside sialicacid and learning ability in well-nourished and malnourished animals andthat these changes persist into adulthood.

Mother's milk is recommended for all infants. However, in some casesbreast feeding is inadequate or unsuccessful for medical reasons or themother chooses not to breast feed. Infant formulas have been developedfor these situations. However, bovine milk which is usually used as thebasis of commercially available infant formulas has a much lower contentof sialylated oligosaccharides than human milk. As the structure ofhuman milk and the functions of the individual components thereofbecomes better understood, it has become apparent that it may bedesirable to improve the sialic acid content of infant formulas based onbovine milk.

There are several known sources of sialic acid including freeN-acetylneuraminic acid, sialyllactose and other sialylatedoligosaccharides, sialic-acid containing gangliosides, and the peptidecaseinoglycomacropeptide. Caseinoglycomacropeptide or CGMP is a largecarbohydrate-rich, hydrophilic peptide which is the C-terminal moiety ofκ casein from which it may be cleaved by enzymatic or acid treatment. InUnited States Patent Application No. 2005/0096295 it is proposed toincrease the sialic acid content of infant formula by including a novelCGMP ingredient having an enhanced concentration of sialic acid. Thisingredient has a sialic acid content of more than 60 mg/g of peptidecompared with 40 to 60 mg/g of peptide in naturally occurring CGMP. Itmay be produced either by fractionation using anion chromatography or bypartial hydrolysis followed by fractionation using anion chromatography.

However, a need remains to provide other ingredients rich in sialic acidwhich can be used to enrich infant formula and other nutritionalcompositions in this important component.

SUMMARY OF THE INVENTION

The present invention provides an oligosaccharide ingredient comprisingglycosylated amino acids and peptides of the general formulaR_(n)Sac_(m) where R is an amino acid residue, Sac is a monosaccharideselected from the group comprising N-acetyl-neuraminic acid, N-acetylgalactosamine and galactose, n has a value between 1 and 10 with theproviso that if n has the value 1 R is a threonine residue or a serineresidue and if n has a value between 2 and 10 the peptide contains atleast one threonine or serine residue, m has a value between 2 and 4 andat least 20 mol % of the ingredient is N-acetyl-neuraminic acid.

This ingredient is a new food grade ingredient rich in sialic acid whichis suitable for addition to nutritional compositions such as infantformula to increase the sialic acid content of the composition.

The invention further extends to a process for producing anoligosaccharide ingredient comprising glycosylated amino acids andpeptides of the general formula RnSacm where R is an amino acid residue,Sac is a monosaccharide selected from the group comprisingN-acetyl-neuraminic acid, N-acetyl galactosamine and galactose, n has avalue between 1 and 10 with the proviso that if n has the value 1 R is athreonine residue or a serine residue and if n has a value between 2 and10 the peptide contains at least one threonine or serine residue, m hasa value between 2 and 4 and at least 20 mol % of the ingredient isN-acetyl-neuraminic acid, comprising the steps of hydrolysingcaseinoglycomacropeptide with an exoprotease and an endoprotease toobtain a mixture of free amino acids and peptides with a chain lengthbetween 2 and 10 and subjecting the hydrolysed mixture to nanofiltrationso as to retain the fraction having a molecular weight between 1000 and2000 Daltons.

The invention also extends to a food product comprising anoligosaccharide ingredient as described above. Optionally the foodproduct is an infant food or formula, but the product may be any food ordrink consumed by babies, infants or adults. Consumption of a foodproduct containing such an oligosaccharide ingredient may improveincorporation of sialic acid into brain gangliosides and thus improverelated neural functions such as neurotransmission.

DETAILED DESCRIPTION OF THE INVENTION

In the present specification, the following words are given a definitionthat must be taken into account when reading and interpreting thedescription, examples and claims.

“CGMP”: caseino-glycomacropeptide, i.e. the glycomacropeptide that iscleaved from bovine κ-casein by the action of the enzyme rennin.

“Infant formula”: foodstuff intended for the complete nutrition ofinfants during the first four to six months of life and for theirpartial nutrition thereafter. The term includes both starter andfollow-on formulas.

“Sialic acid”: N-acetylneuraminic acid.

The invention provides an oligosaccharide ingredient which comprisesglycosylated amino acids and peptides of the general formulaR_(n)Sac_(m) where R is an amino acid residue, Sac is a monosaccharideselected from the group comprising N-acetyl-neuraminic acid, N-acetylgalactosamine and galactose, n has a value between 1 and 10 with theproviso that if n has the value 1 R is a threonine residue or a serineresidue and if n has a value between 2 and 10 the peptide contains atleast one threonine or serine residue, m has a value between 2 and 4 andat least 20 mol % of the ingredient is N-acetyl-neuraminic acid as wellas infant or adult food products comprising such an oligosaccharideingredient.

Preferably n has a value between 1 and 3 and m has a value of 3 or 4.

The ingredient contains at least 20 mol % sialic acid as part of asaccharide chain linked to the hydroxyl group of threonine or serine.The sialic acid may form part of the chain or may itself be asubstituent of a monosaccharide unit in the chain. Thus, theoligosaccharide ingredient may contain the following monosaccharides:—

Compound mol % N-acetyl galactosamine (GalNAc) 10-25 galactose (Gal)10-25 N-acetyl-neuraminic acid (NeuAc) 50-20

The oligosaccharide ingredient may contain from 15 to 50 mol % of amixture of serine and/or threonine.

The oligosaccharide ingredient may contain the following glycosylatedamino acids or peptides:—

NeuAc-α-2,3-Gal-β-1,3-(NeuAc-α-2,6-)-GalNAc-R_(n)

NeuAc-α-2,3-Gal-β-1,3-GalNAc-R_(n)

Gal-β-1,3-(NeuAc-α-2,6-)-GalNAc-R_(n)

Gal-β-1,3-GalNAc-R_(n)

The oligosaccharide ingredient of the invention may be produced by thehydrolysis of CGMP using an exoprotease and an endoprotease to obtain amixture of free amino acids and peptides with a chain length between 2and 10 and subjecting the hydrolysed mixture to nanofiltration so as toretain the fraction having a molecular weight between 1000 and 2000Daltons.

CGGMP itself is a by-product of cheese-making in which whole milk istreated with the enzyme rennin to precipitate the casein. In thisprocess, CGMP is cleaved from κ casein and remains in solution with thewhey proteins. This product is known as sweet whey. The CGMP may beseparated from the whey proteins by any process known in the art. Asuitable process is described in European Patent No. 986312.

The hydrolysis may be carried out using any suitable combination ofenzymes. One example of a commercially available enzyme system with exo-and endo-protease activity is the product sold as Flavourzyme® by theNovozymes division of Novo Nordisk.

The product of the hydrolysis process is a mixture of free amino acids,glycosylated threonine and serine, glycosylated peptides containing atleast one threonine or serine residue and unglycosylated peptides, thepeptides having a chain of 2 to 10 amino acid residues. This mixture issubject to nanofiltration to separate and retain the fraction having amolecular weight in the range from 1000 to 2000 Daltons. This fractionwill comprise the oligosaccharide ingredient of the invention.

In a preferred aspect of the invention, the oligosaccharide ingredientdescribed above is incorporated into a food product. In the context ofthe present invention, the term “food product” is intended to encompassany consumable matter. Hence, it may be a product intended forconsumption by humans, in particular infant formula, follow-up formula,baby food such as infant cereals and the like. In particular, theoligosaccharide ingredient of the invention can be incorporated intoinfant formulas, growing up milks, and dehydrated milk or cerealmixtures.

The food product may be prepared in any suitable manner known in the artaccording to the type of product and the oligosaccharide ingredient ofthe invention may be added to the product at an appropriate stage in themanufacturing process. For example, an infant formula may be prepared byblending together the protein source, any carbohydrates other thanlactose and the fat source in appropriate proportions. Emulsifiers maybe added if desired. Vitamins and minerals may be added at this pointbut are usually added later to avoid thermal degradation. Any lipophilicvitamins, emulsifiers and the like may be dissolved into the fat sourceprior to blending. Water, preferably water which has been subjected toreverse osmosis, may then be mixed in to form a liquid mixture.

The liquid mixture may then be thermally treated to reduce bacterialloads. For example, the liquid mixture may be rapidly heated to atemperature in the range of about 80° C. to about 110° C. for about 5seconds to about 5 minutes. This may be carried out by steam injectionor by heat exchanger, e.g. a plate heat exchanger.

The liquid mixture may then be cooled to about 60° C. to about 85° C.,for example by flash cooling. The liquid mixture may then behomogenised, for example in two stages at about 7 MPa to about 40 MPa inthe first stage and about 2 MPa to about 14 MPa in the second stage. Thehomogenised mixture may then be further cooled to add any heat sensitivecomponents such as vitamins and minerals. The pH and solids content ofthe homogenised mixture is conveniently standardised at this point.

The homogenised mixture is transferred to a suitable drying apparatus,such as a spray drier or freeze drier, and converted to powder. Thepowder should have a moisture content of less than about 5% by weight.

The oligosaccharide ingredient of the invention may be added directly toinfant formula by dry mixing or, if in liquid for, at the blending stagediscussed above. In both cases, it will be appreciated that addition ofthe ingredient entails the simultaneous addition of a quantity ofamino-nitrogen and it may therefore be necessary to adjust the proteincontent of the infant formula or other product to which the ingredientis being added accordingly as will be evident to one skilled in the art.The permissible protein content and amino acid profile of infantformulas are specified in legislation such as European CommissionDirective 91/321/EEC of 14 May 1991 and the person skilled in the artwill easily be able to adjust the protein content and amino acid profileof the infant formula to take account of the additional amino-nitrogenintroduced by the ingredient of the invention as well as the additionalthreonine and serine content.

The final concentration of the oligosaccharide ingredient in the baby orinfant food product or formula may be from 0.2 to 4.0%, preferably 0.5to 2.0% by weight of dry matter. However, these amounts should not beconsidered as limitative and should be adapted to the target population,for example based on the weight and age or health of the baby or infant.Preferably, the formula or feed containing the oligosaccharideingredient of the invention is fed to the baby at every feed.

Although it is preferred to supplement food products specificallytargeted towards infant or baby nutrition, it may be beneficial tosupplement food products not specifically targeted, or targeted to theadult population. For example, the oligosaccharide ingredient of theinvention can be incorporated into healthcare nutrition products andnutritional products for the elderly. Such food products may includemilk, yoghurt, curd, cheese, fermented milks, milk-based fermentedproducts, ice-creams, fermented cereal based products, or milk-basedproducts, among others. In addition to the oligosaccharide ingredient ofthe invention, a food product such as an infant formula may comprise oneor more further oligosaccharides which are added separately.

The invention will now be illustrated by reference to the followingexample.

Example 1

An example of the composition of an infant formula containing aoligosaccharide ingredient according to the present invention is givenbelow.

Nutrient per 100 kcal per litre Energy (kcal) 100 670 Protein (g) 1.8312.3 Fat (g) 5.3 35.7 Linoleic acid (g) 0.79 5.3 α-Linolenic acid (mg)101 675 Lactose (g) 11.2 74.7 OS ingredient (g) 0.15 1.0 Minerals (g)0.37 2.5 Na (mg) 23 150 K (mg) 89 590 Cl (mg) 64 430 Ca (mg) 62 410 P(mg) 31 210 Mg (mg) 7 50 Mn (μg) 8 50 Se (μg) 2 13 Vitamin A (μg RE) 105700 Vitamin D (μg) 1.5 10 Vitamin E (mg TE) 0.8 5.4 Vitamin K1 (μg) 8 54Vitamin C (mg) 10 67 Vitamin B1 (mg) 0.07 0.47 Vitamin B2 (mg) 0.15 1.0Niacin (mg) 1 6.7 Vitamin B6 (mg) 0.075 0.50 Folic acid (μg) 9 60Pantothenic acid (mg) 0.45 3 Vitamin B12 (μg) 0.3 2 Biotin (μg) 2.2 15Choline (mg) 10 67 Fe (mg) 1.2 8 I (μg) 15 100 Cu (mg) 0.06 0.4 Zn (mg)0.75 5

Example 2

50 g of caseinoglycomacropeptide (Biopure GMP, Davisco, USA) weredissolved at 10% (w/v) in 500 ml deionized water. The solution heated at40° C. for 30 minutes while stirring gently and recirculated through alabscale tangential flow filtration system (Millipore, USA) with onePellicon 1000 Da filter cassette (0.1 m²) at a feed pressure of 1.2 barand no retentate pressure.

Thereafter, an aminopeptidase from Aspergillus oryzae (Flavourzyme 1000L containing 1000 aminopeptidase units per gram (LAPU/g), Novozymes, DK)was added at 3500 LAPU per 50 g protein. After about 1 hr, diafiltrationwas started by increasing the retentate pressure to 3 bars with a feedpressure of 3 bars. After 5 hrs the filtration was stopped and theretentate was collected. The retentate and the 7 permeate fractions thatwere collected during the process were lyophilized. Thereafter, drymatter (DM) and sialic acid content of each fraction was determined.Sialic acid content was measured after mild acid hydrolysis andlabelling using the DMB (1,2-Diamino-4,5-methylenedioxybenzenedihydrochloride) method and analysis on an HPLC equipped with a ShodexC18 reverse phase column and a fluorescence detector using authenticNeuAc as external standards. Table 1 shows the mass balance and sialicacid content in starting material, permeate fractions and retentate.

TABLE 1 mass (g) NeuAc(% of DM) yield (%) enrichment starting material50 7.5 100 1.00 permeate 1 4.32 0.2 8.64 0.03 permeate 2 3.1 0.2 6.20.02 permeate 3 6.1 0.3 12.2 0.04 permeate 4 4.29 1.4 8.58 0.18 permeate5 3.56 0.8 7.12 0.10 permeate 6 2.8 0.4 5.6 0.05 permeate 7 2.94 0.35.88 0.04 retentate 22.6 14.4 45.2 1.92

Galactose and N-acetyl-galactosamine content in the retentate weredetermined after acid hydrolysis using an HPAEC equipped with a DionexCarboPac PA1 analytical column and a pulsed amperometry detector.Briefly, retentate was dissolved in water and tri-fluoroacetic acid wasadded to a final concentration of 2M. The solution was heated for 3 hrat 100° C., dried under a N-stream and redissolved with water. Afterfiltration through a 22 um particle filter the sample was analysed.Quantification was done with authentic galactose and galactosamine(N-acetylgalactosamine becomes galactosamine during the acid treatment)as external standards. Threonine was quantified by HPLC after acidhydrolysis with 6N HCl.

The final dried retentate product contained 29.6 wt %(NeuAc)₂₋₃-Gal-GalNAc-Thr with the likely structureNeuAc-α-2,3-Gal-β-1,3-(NeuAc-α-2,6-)-GalNAc-threonine/peptide with 30mol % sialic acid, 15 mol % galactose, 13 mol % N-acetyl galactosamineand 42 mol % threonine.

The invention claimed is:
 1. An oligosaccharide ingredient comprisingglycosylated amino acids and peptides of a general formula R_(n)Sac_(m)where R is an amino acid residue, Sac is a monosaccharide selected fromthe group comprising N-acetyl-neuraminic acid, N-acetyl galactosamineand galactose, n has a value between 1 and 10 with the proviso that if nis 1 R is selected from the group consisting of a threonine residue anda serine residue and if n has a value between 2 and 10 the peptidecontains at least one residue selected from the group consisting ofthreonine and serine residue, m has a value between 2 and 4, theoligosaccharide ingredient comprising 10 to 25 mol % N-acetylgalactosamine, 10 to 25 mol % galactose, 20 to 50 mol %N-acetyl-neuraminic acid and 15 to 50 mol % threonine or serine or amixture thereof, the oligosaccharide ingredient comprising aglycosylated amino acid or peptide selected from the group consisting ofNeuAc-α-2,3-Gal-β-1,3-(NeuAc-α-2,6-)-GalNAc-R_(n),NeuAc-α-2,3-Gal-β-1,3-GalNAc-R_(n),Gal-β-1,3-(NeuAc-α-2,6-)-GalNAc-R_(n), and Gal-β-1,3-GalNAc-R_(n).
 2. Anoligosaccharide ingredient as claimed in claim 1 wherein n has a valuebetween 1 and
 3. 3. An oligosaccharide ingredient as claimed in claim 1wherein m has a value of 2 or
 3. 4. A food product comprising anoligosaccharide ingredient comprising glycosylated amino acids andpeptides of a general formula R_(n)Sac_(m) where R is an amino acidresidue, Sac is a monosaccharide selected from the group comprisingN-acetyl-neuraminic acid, N-acetyl galactosamine and galactose, n has avalue between 1 and 10 with the proviso that if n is 1 R is selectedfrom the group consisting of a threonine residue and a serine residueand if n has a value between 2 and 10 the peptide contains at least oneresidue selected from the group consisting of threonine and serineresidue, m has a value between 2 and 4 and at least 20 mol % of theingredient is N-acetyl-neuraminic acid, the oligosaccharide ingredientcomprising 10 to 25 mol % N-acetyl galactosamine, 10 to 25 mol %galactose, 20 to 50 mol % N-acetyl-neuraminic acid and 15 to 50 mol %threonine or serine or a mixture thereof, the oligosaccharide ingredientcomprising a glycosylated amino acid or peptide selected from the groupconsisting of NeuAc-α-2,3-Gal-β-1,3-(NeuAc-α-2,6-)-GalNAc-R_(n),NeuAc-α-2,3-Gal-β-1,3-GalNAc-R_(n),Gal-β-1,3-(NeuAc-α-2,6-)-GalNAc-R_(n), and Gal-β-1,3-GalNAc-R_(n).
 5. Afood product as claimed in claim 4 which is an infant formula.
 6. A foodproduct as claimed in claim 4 comprising from 0.2 to 4.0% by weight ofthe oligosaccharide ingredient.
 7. An oligosaccharide ingredient asclaimed in claim 1 wherein the oligosaccharide ingredient has at leastone glycosylated amino acid selected from the group consisting ofNeuAc-α-2,3-Gal-β-1,3-(NeuAc-α-2,6-)-GalNAc-R_(n) andGal-β-1,3-(NeuAc-α-2,6-)-GalNAc-R_(n).
 8. A food product as claimed inclaim 4, wherein the oligosaccharide ingredient comprises at least oneglycosylated amino acid selected from the group consisting ofNeuAc-α-2,3-Gal-β-1,3-(NeuAc-α-2,6-)-GalNAc-R_(n) andGal-β-1,3-(NeuAc-α-2,6-)-GalNAc-R_(n).